# Patent

## Title
Haemophilus parasuis polypeptides and methods of use

## Abstract

The present invention provides isolated polypeptides having oligopeptide permease activity and an amino acid sequence that has at least 80% identity with a Haemophilus parasuis OppA polypeptide. Also provided by the present invention are isolated polynucleotides that encode the polypeptides described herein, and antibody that specifically binds a polypeptide described herein. The present invention further provides genetically modified microbes, such as attenuated Haemophilus parasuis strains and other microbes that express polypeptides described herein. Also included are methods for using the polypeptides, polynucleotides, antibody, and genetically modified microbes.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a U.S. National Stage Application of International Application No. PCT/US2010/055425, titled HAEMOPHILUS PARASUIS POLYPEPTIDES AND METHODS OF USE, filed on Nov. 4, 2010, published in the English language on May 12, 2011 as International Publication No. WO 2011/056954 A2, which claims the benefit of U.S. Provisional Application Ser. No. 61/257,921, filed Nov. 4, 2009, and U.S. Provisional Application Ser. No. 61/314,468, filed Mar. 16, 2010, each of which is incorporated by reference herein.


BACKGROUND
Haemophilus parasuis is an important cause of mortality in nursery pigs (Oliveira and Pijoan, 2004, Vet. Microbiol., 99(1):1-12). It is a commensal organism that colonizes virtually 100% of swine herds worldwide, and a frequent opportunistic pathogen during concurrent viral infections and stressful events (Oliveira et al., 2004, Safety of Controlled exposure to Haemophilus parasuis: the role of sow vaccination and PRRS virus infection. Proceedings of the 18th International Pig Veterinary Congress, Hamburg, Germany, p. 189), causing up to 60% mortality in naïve populations.
Protection against H. parasuis systemic infection is associated with the presence of antibodies against this pathogen (Solano-Aguilar et al., 1999, Am. J. Vet. Res., 60(1):81-87). The high susceptibility of colostrum-deprived pigs to H. parasuis systemic infection corroborates the importance of maternal antibodies as a major defense mechanism against this agent (Oliveira et al., 2003, Can. J. Vet. Res., 67(2):146-150). Commercial and autogenous vaccines currently available for H. parasuis generally do not provide complete cross-protection among different strains and serotypes due to extensive genotypic and phenotypic variation between different H. parasuis strains (Oliveira et al., 2003, Am. J. Vet. Res., 64(4):435-442, Oliveira et al., 2004, J. Swine Health and Production, 12(3):123-128). This is a major limitation to preventing nursery mortality against this pathogen. Antibiotics can be useful in controlling H. parasuis outbreaks; however, like other modern food-animal based industries, the swine industry is rapidly reducing the use of antibiotics to control mortality.
SUMMARY OF THE INVENTION
Haemophilus parasuis continues to be one of the most important causes of nursery mortality in modern swine production. Challenges in H. parasuis control include the lack of a reliable serological test to characterize the development of antibodies in sows and piglets, and the absence of vaccine that acts to suppress disease caused by H. parasuis. As described herein, a highly immunogenic and species-specific protein has been identified in H. parasuis. This protein was not only present in H. parasuis strains causing outbreaks, but was also present in reference strains representing 13 different serotypes. The convalescent serum that detected this protein did not detect a similar antigen in 10 other closely related bacterial swine pathogens tested, confirming that this protein was highly specific for H. parasuis. The highly immunogenic protein was identified as the oligopeptide permease A (OppA), which is a component of the ATP-binding cassette (ABC) transporter system in H. parasuis. Testing of serum samples obtained from non-clinical, clinical, and convalescent pigs suggested that colonized healthy pigs do not respond to this protein, whereas survivor pigs generated high titers against H. parasuis OppA.
The present invention provides isolated polynucleotides. The polynucleotides encode a polypeptide having oligopeptide permease activity, or a fragment thereof. In one embodiment the amino acid sequence of the polypeptide has at least 80% identity with SEQ ID NO:2. In another embodiment, the nucleotide sequence of the polynucleotide has at least 80% identity with SEQ ID NO:1. The full complement of the polynucleotides are also included in the present invention. The polynucleotides may include a heterologous polynucleotide, such as a regulatory sequence or a vector.
Also provided by the present invention are isolated polypeptides. The polypeptides have oligopeptide permease activity, and an amino acid sequence that has at least 80% identity with SEQ ID NO:2. Also included in the present invention are fragments of the polypeptides, and such fragments may have oligopeptide permease activity. The polypeptides may include a heterologous polypeptide.
The present invention further provides genetically modified microbes. The genetically modified microbes may include an exogenous polynucleotide, wherein the exogenous polynucleotide is a polynucleotide of the present invention. A genetically modified microbe may be E. coli. A genetically modified microbe of the present invention may be a Haemophilus parasuis. Such a genetically modified Haemophilus parasuis may include an oppA coding region that contains a mutation, such as a point mutation, a deletion, an insertion, or a combination thereof. The polypeptide encoded by the oppA coding region may have oligopeptide permease activity, and in other embodiments may not have oligopeptide permease activity. In one embodiment the oppA coding region does not encode a polypeptide. In some embodiments the genetically modified Haemophilus parasuis has decreased virulence in a pig.
Also included in the present invention is antibody that specifically binds a polypeptide having SEQ ID NO:2, wherein the antibody does not bind to a polypeptide that has an amino acid sequence SEQ ID NO:4. The antibody may be present in a composition that includes a pharmaceutically acceptable carrier. In one embodiment the antibody is a monoclonal antibody. The present invention also provides methods for making an antibody. The method may include administering to an animal a polypeptide in an amount effective to cause the production of an antibody specific for the polypeptide, where the polypeptide includes an amino acid sequence having at least 80% identity with SEQ ID NO:2 and has oligopeptide permease activity. The method may include isolating the antibody.
The present invention provides methods for detecting antibody. The method may include mixing a biological sample from an animal with a polypeptide under conditions suitable for formation of a polypeptide:antigen complex between antibody present in the biological sample and the polypeptide, where the polypeptide includes an amino acid sequence having at least 80% identity with SEQ ID NO:2 and has oligopeptide permease activity. The polypeptide may be bound to a surface. The method may include detecting the complex, where the presence of the complex indicates the animal has antibody that specifically binds to the polypeptide. Detecting the complex may include contacting the antibody with a secondary antibody, such as a secondary antibody that includes a detectable label. The presence of the complex may indicate the animal has been infected with Haemophilus parasuis. The biological sample may include serum, oral fluid, colostrum, lung lavage, bronchial lavage, tracheal lavage, or nasal lavage. The biological sample may be from a pig.
In another embodiment, the method may include mixing a biological sample from an animal with an antibody under conditions suitable for formation of a polypeptide:antigen complex between a polypeptide present in the biological sample and the antibody, where the antibody specifically binds a polypeptide having an amino acid SEQ ID NO:2. The antibody may be bound to a surface. The method may include detecting the complex, where the presence of the complex indicates the biological sample includes a Haemophilus parasuis OppA polypeptide. Detecting the complex may include contacting the polypeptide with a secondary antibody, such as a secondary antibody that includes a detectable label. The presence of the complex may indicate the animal has been infected with Haemophilus parasuis. The biological sample may include serum, oral fluid, colostrum, lung lavage, bronchial lavage, tracheal lavage, or nasal lavage. The biological sample may be from a pig.
Further provided by the present invention are methods for detecting the presence of a Haemophilus parasuis. Such methods may include amplifying a target polynucleotide by contacting the target polynucleotide with a primer pair under suitable conditions to result in an amplified product, wherein the primer pair amplifies a target nucleotide including SEQ ID NO:1 and does not amplify a target nucleotide including SEQ ID NO:3, and detecting the amplified product, wherein the presence of the amplified product is indicative of the presence of a Haemophilus parasuis. The target polynucleotide may be present in a biological sample.
The present invention also provides methods for treating an animal at risk of infection with a Haemophilus parasuis. The methods may include administering to the animal a composition that includes an attenuated Haemophilus parasuis, where the attenuated Haemophilus parasuis includes a mutation in an oppA coding region or an operably linked regulatory region, and wherein the Haemophilus parasuis has decreased virulence in a pig when compared to the same Haemophilus parasuis having a wild-type oppA coding region.
Also provided by the present invention are methods for treating an animal at risk of infection with a Haemophilus parasuis. The methods may include administering to the animal a composition that includes a polypeptide or a fragment thereof, wherein the amino acid sequence of the polypeptide has at least 80% identity to the amino acid sequence of SEQ ID NO: 2, and wherein the polypeptide has oligopeptide permease activity.
As used herein, the term “polynucleotide” refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxynucleotides, and includes both double- and single-stranded RNA and DNA. A polynucleotide can be obtained directly from a natural source, or can be prepared with the aid of recombinant, enzymatic, or chemical techniques. A polynucleotide can be linear or circular in topology. A polynucleotide may be, for example, a portion of a vector, such as an expression or cloning vector, or a fragment. A polynucleotide may include nucleotide sequences having different functions, including, for instance, coding regions, and non-coding regions such as regulatory regions.
As used herein, the terms “coding region” and “coding sequence” are used interchangeably and refer to a nucleotide sequence that encodes a polypeptide and, when placed under the control of appropriate regulatory sequences expresses the encoded polypeptide. The boundaries of a coding region are generally determined by a translation start codon at its 5′ end and a translation stop codon at its 3′ end. A “regulatory sequence” is a nucleotide sequence that regulates expression of a coding sequence to which it is operably linked. Non-limiting examples of regulatory sequences include promoters, enhancers, transcription initiation sites, translation start sites, translation stop sites, and transcription terminators. The term “operably linked” refers to a juxtaposition of components such that they are in a relationship permitting them to function in their intended manner. A regulatory sequence is “operably linked” to a coding region when it is joined in such a way that expression of the coding region is achieved under conditions compatible with the regulatory sequence.
A polynucleotide that includes a coding region may include heterologous nucleotides that flank one or both sides of the coding region. As used herein, “heterologous nucleotides” refer to nucleotides that are not normally present flanking a coding region that is present in a wild-type cell. For instance, a coding region present in a wild-type microbe and encoding a polypeptide is flanked by homologous sequences, and any other nucleotide sequence flanking the coding region is considered to be heterologous. Examples of heterologous nucleotides include, but are not limited to regulatory sequences. Typically, heterologous nucleotides are present in a polynucleotide of the present invention through the use of standard genetic and/or recombinant methodologies well known to one skilled in the art. A polynucleotide of the present invention may be included in a suitable vector. The presence of heterologous nucleotides flanking one or both sides of a polynucleotide described herein result from human manipulation.
As used herein, an “exogenous polynucleotide” refers to a polynucleotide that is not normally or naturally found in a microbe. As used herein, the term “endogenous polynucleotide” refers to a polynucleotide that is normally or naturally found in a microbe. An “endogenous polynucleotide” is also referred to as a “native polynucleotide.”
The terms “complement” and “complementary” as used herein, refer to the ability of two single stranded polynucleotides to base pair with each other, where an adenine on one strand of a polynucleotide will base pair to a thymine or uracil on a strand of a second polynucleotide and a cytosine on one strand of a polynucleotide will base pair to a guanine on a strand of a second polynucleotide. Two polynucleotides are complementary to each other when a nucleotide sequence in one polynucleotide can base pair with a nucleotide sequence in a second polynucleotide. For instance, 5′-ATGC and 5′-GCAT are complementary. The term “substantial complement” and cognates thereof as used herein, refer to a polynucleotide that is capable of selectively hybridizing to a specified polynucleotide under stringent hybridization conditions. Stringent hybridization can take place under a number of pH, salt and temperature conditions. The pH can vary from 6 to 9, preferably 6.8 to 8.5. The salt concentration can vary from 0.15 M sodium to 0.9 M sodium, and other cations can be used as long as the ionic strength is equivalent to that specified for sodium. The temperature of the hybridization reaction can vary from 30° C. to 80° C., preferably from 45° C. to 70° C. Additionally, other compounds can be added to a hybridization reaction to promote specific hybridization at lower temperatures, such as at or approaching room temperature. Among the compounds contemplated for lowering the temperature requirements is formamide. Thus, a polynucleotide is typically substantially complementary to a second polynucleotide if hybridization occurs between the polynucleotide and the second polynucleotide. As used herein, “specific hybridization” refers to hybridization between two polynucleotides under stringent hybridization conditions.
As used herein, the term “polypeptide” refers broadly to a polymer of two or more amino acids joined together by peptide bonds. The term “polypeptide” also includes molecules which contain more than one polypeptide joined by a disulfide bond, or complexes of polypeptides that are joined together, covalently or noncovalently, as multimers (e.g., dimers, tetramers). Thus, the terms peptide, oligopeptide, enzyme, and protein are all included within the definition of polypeptide and these terms are used interchangeably. It should be understood that these terms do not connote a specific length of a polymer of amino acids, nor are they intended to imply or distinguish whether the polypeptide is produced using recombinant techniques, chemical or enzymatic synthesis, or is naturally occurring.
As used herein, “oligopeptide permease activity” refers to activity of OppA polypeptides described herein. OppA polypeptides are one component of oligopeptide transport systems of the ATP-binding cassette (ABC) family of transporters (Monnet, 2003, Cell. Mol. Life. Sci., 60:2100-2114). Such systems include five parts: an oligopeptide-binding polypeptide which binds substrates, two transmembrane polypeptides, and two polypeptides that hydrolize ATP. An OppA polypeptide described herein binds oligopeptides, typically between two and five amino acids in length, and pass the bound polypeptide to the other components of the oligopeptide transport system for internalization. Methods for determining whether a OppA polypeptide has oligopeptide permase activity are well known to the skilled person.
As used herein, an “isolated” substance is one that has been removed from its natural environment, produced using recombinant techniques, or chemically or enzymatically synthesized. For instance, a polypeptide or a polynucleotide can be isolated. Preferably, a substance is purified, i.e., is at least 60% free, preferably at least 75% free, and most preferably at least 90% free from other components with which they are naturally associated.
As used herein, “identity” refers to sequence similarity between two polypeptides or two polynucleotides. The sequence similarity between two polypeptides is determined by aligning the residues of the two polypeptides (e.g., a candidate amino acid sequence and a reference amino acid sequence, such as SEQ ID NO:2) to optimize the number of identical amino acids along the lengths of their sequences; gaps in either or both sequences are permitted in making the alignment in order to optimize the number of shared amino acids, although the amino acids in each sequence must nonetheless remain in their proper order. The sequence similarity is typically at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity. Sequence similarity may be determined, for example, using sequence techniques such as the BESTFIT algorithm in the GCG package (Madison Wis.), or the Blastp program of the BLAST search algorithm, available through the World Wide Web, for instance at the interne site maintained by the National Center for Biotechnology Information, National Institutes of Health. Preferably, sequence similarity between two amino acid sequences is determined using the Blastp program of the BLAST search algorithm. Preferably, the default values for all Blastp search parameters are used. In the comparison of two amino acid sequences using the BLAST search algorithm, structural similarity is referred to as “identities.”
The sequence similarity between two polynucleotides is determined by aligning the residues of the two polynucleotides (e.g., a candidate nucleotide sequence and a reference nucleotide sequence, such as the complement of SEQ ID NO:1) to optimize the number of identical nucleotides along the lengths of their sequences; gaps in either or both sequences are permitted in making the alignment in order to optimize the number of shared nucleotides, although the nucleotides in each sequence must nonetheless remain in their proper order. The sequence similarity is typically at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity. Sequence similarity may be determined, for example, using sequence techniques such as GCG FastA (Genetics Computer Group, Madison, Wis.), MacVector 4.5 (Kodak/IBI software package) or other suitable sequencing programs or methods known in the art. Preferably, sequence similarity between two nucleotide sequences is determined using the Blastn program of the BLAST search algorithm, available through the World Wide Web, for instance at the internet site maintained by the National Center for Biotechnology Information, National Institutes of Health. Preferably, the default values for all Blastn search parameters are used. In the comparison of two nucleotide sequences using the BLAST search algorithm, sequence similarity is referred to as “identities.”
Conditions that “allow” an event to occur or conditions that are “suitable” for an event to occur, such as an enzymatic reaction, or “suitable” conditions are conditions that do not prevent such events from occurring. Thus, these conditions permit, enhance, facilitate, and/or are conducive to the event. Such conditions, known in the art and described herein, may depend upon, for example, the antibody being used.
As used herein, a polypeptide “fragment” may retain at least some of the activity of the corresponding native polypeptide. Examples of fragments of polypeptides described herein include, but are not limited to, proteolytic fragments and deletion fragments.
As used herein, “genetically modified microbe” refers to a microbe that has been altered by human intervention. For example, a microbe is a genetically modified microbe by virtue of introduction into a suitable microbe of an exogenous polynucleotide that is foreign to the microbe. “Genetically modified microbe” also refers to a microbe that has been genetically manipulated such that endogenous nucleotides have been altered. For example, a microbe is a genetically modified microbe by virtue of introduction into a suitable microbe of an alteration of endogenous nucleotides. For instance, an endogenous coding region could be deleted or modified to include a mutation. Such mutations may result in a polypeptide having a different amino acid sequence than was encoded by the endogenous polynucleotide, or not being expressed. Another example of a genetically modified microbe is one having an altered regulatory sequence, such as a promoter, to result in increased or decreased expression of an operably linked endogenous coding region.
As used herein, an antibody that can “specifically bind” or is “specific for” a polypeptide is an antibody that interacts only with an epitope of the antigen that induced the synthesis of the antibody, or interacts with a structurally related epitope.
The term “and/or” means one or all of the listed elements or a combination of any two or more of the listed elements.
The words “preferred” and “preferably” refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the invention.
The terms “comprises” and variations thereof do not have a limiting meaning where these terms appear in the description and claims.
Unless otherwise specified, “a,” “an,” “the,” and “at least one” are used interchangeably and mean one or more than one.
Also herein, the recitations of numerical ranges by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).
For any method disclosed herein that includes discrete steps, the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously.
The summary of the present invention presented below is not intended to describe each disclosed embodiment or every implementation of the present invention. The description that follows more particularly exemplifies illustrative embodiments. In several places throughout the application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1. Detection of the immunogenic species-specific antigen in 13 Haemophilus parasuis reference strains by western blot of whole cell protein profiles. Serotypes are indicated above each well; M, molecular weight marker.
FIG. 2. Detection of the immunogenic species-specific antigen in 7 Haemophilus parasuis field strains obtained from an outbreak from brain (BR), pleura (PL), and pericardium (PC) and 3 Haemophilus parasuis references strains (Ser3, serotype 3; Ser5, serotype 5; and Ser12, serotype 12) by western blot of whole cell protein profiles. M, molecular weight marker.
FIG. 3. Specificity testing using serum from a piglet obtained prior to colostrum suckling. M, molecular weight marker.
FIG. 4. Specificity testing by western blot of whole cell protein profiles using serum from a convalescent pig that survived a H. parasuis outbreak. AP, Actinobacillus porcinus; AI, Actinobacillus indolicus; AM, Actinobacillus minor; APP, Actinobacillus pleuropneumoniae; AS, Actinobacillus suis; SS, Streptococcus suis; BB, Bordetella bronchiseptica; PM, Pasteurella multocida; EC, Escherichia coli; SC, Salmonella choleraesuis; BR7, a Haemophilus parasuis strains isolated from brain; and Ser 5, reference strain for serotype 5. M, molecular weight marker.
FIG. 5. SDS-PAGE showing 52 kDa protein identified by convalescent sera (western blot) and selected for further identification by sequencing. Ser5, serotype 5; M, molecular weight marker.
FIG. 6. Oligopeptide permease operon in Haemophilus parasuis. The protein identified as described herein is encoded by the OppA segment of this operon.
FIG. 7. Anti-OppA antibodies sample to positive (S/P) ratios obtained for serum samples collected from 10 pigs that survived a Haemophilus parasuis outbreak.
FIG. 8. Anti-OppA antibodies sample to positive (S/P) ratios obtained for serum samples collected from 5 (A) and 19 (B) day old piglets.
FIG. 9. (A) Anti-OppA antibodies sample to positive (S/P) ratios obtained for 26 piglets at 3 weeks of age. All pigs lacked antibodies against Haemophilus parasuis OppA. This profile coincides with decay of maternal immunity and absence of active immunity in this population. (B) Anti-OppA S/P ratios obtained for 30 pigs at 8 weeks of age in the same population. All pigs had S/P ratios above 0.1, indicating the development of active immune response following systemic contact with Haemophilus parasuis after surviving the peak of nursery mortality. Cross-sectional sampling.
FIG. 10. Anti OppA S/P ratios obtained from 26 pigs from a naturally exposed, colonized, non-vaccinated population at 1 (A) and 9 (B) weeks of age. Some maternal immunity was evident at 1 week-old, whereas seroconversion was evident at 9 weeks of age, after the peak of Haemophilus parasuis-related mortality. Longitudinal sampling.
FIG. 11. A. Full length alignment between an OppA polypeptide of the present invention (SEQ ID NO:2, top sequence) and an Actinobacillus pleuropneumoniae oligopeptide permease (SEQ ID NO:4, bottom sequence). “:” refers to identity between the two sequences. B. Nucleotide sequence encoding SEQ ID NO:4.
FIG. 12. Full length alignment between an OppA polypeptide of the present invention (SEQ ID NO:2, top sequence), an Actinobacillus minor oligopeptide permease (SEQ ID NO:5, second sequence), an Actinobacillus pleuropneumoniae oligopeptide permease (SEQ ID NO:4, third sequence), and a Mannheimia haemolytica oligopeptide permease (SEQ ID NO:6, fourth sequence). “*” refers to identity between the four sequences.
FIG. 13. Polypeptides expressed by subclones. The polyhistidine is underlined and the protease cleavage sites (ENLYFQG, SEQ ID NO:10, and DDDDK, SEQ ID NO:11) are shaded.
FIG. 14. A nucleotide sequence (SEQ ID NO:9) encoding SEQ ID NO:2.



DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
The present invention includes isolated polypeptides having oligopeptide permease activity. A polypeptide having oligopeptide permease activity is referred to herein as an OppA polypeptide. An example of an OppA polypeptide is depicted at SEQ ID NO:2. Other examples of OppA polypeptides of the present invention include those having sequence similarity with the amino acid sequence of SEQ ID NO:2. An OppA polypeptide having sequence similarity with the amino acid sequence of SEQ ID NO:2 has oligopeptide permease activity. An OppA polypeptide may be isolated from a microbe, such as a member of the genera Pasteurellaceae, such as a Haemophilus parasuis. The H. parasuis may be of any serotype, including, but not limited to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, or may be non-typable. An OppA polypeptide may be produced using recombinant techniques, or chemically or enzymatically synthesized using routine methods.
The amino acid sequence of an OppA polypeptide having sequence similarity to SEQ ID NO:2 may include conservative substitutions of amino acids present in SEQ ID NO:2. A conservative substitution is typically the substitution of one amino acid for another that is a member of the same class. For example, it is well known in the art of protein biochemistry that an amino acid belonging to a grouping of amino acids having a particular size or characteristic (such as charge, hydrophobicity, and/or hydrophilicity) may generally be substituted for another amino acid without substantially altering the secondary and/or tertiary structure of a polypeptide. Conservative amino acid substitutions can result from exchange of amino acids residues from within one of the following classes of residues: Class I: Gly, Ala, Val, Leu, and Ile (representing aliphatic side chains); Class II: Gly, Ala, Val, Leu, Ile, Ser, and Thr (representing aliphatic and aliphatic hydroxyl side chains); Class III: Tyr, Ser, and Thr (representing hydroxyl side chains); Class IV: Cys and Met (representing sulfur-containing side chains); Class V: Glu, Asp, Asn and Gln (carboxyl or amide group containing side chains); Class VI: His, Arg and Lys (representing basic side chains); Class VII: Gly, Ala, Pro, Trp, Tyr, Ile, Val, Leu, Phe and Met (representing hydrophobic side chains); Class VIII: Phe, Trp, and Tyr (representing aromatic side chains); and Class IX: Asn and Gln (representing amide side chains). The classes are not limited to naturally occurring amino acids, but also include artificial amino acids, such as beta or gamma amino acids and those containing non-natural side chains, and/or other similar monomers such as hydroxyacids. SEQ ID NO:2 is shown in FIG. 12 in a multiple protein alignment with three other proteins having oligopeptide permease activity. Identical amino acids between all four are marked with a “*.” Significant domains of conserved amino acids are present, and the skilled person could predict which amino acids of SEQ ID NO:2 could be varied without losing activity. The structural basis for peptide binding by OppA polypeptides is also known (Tame et al., 1994, Science, 264:1578-1581, and Tame et al., 1995, Structure, 3:1395-1406).
Guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie et al. (1990, Science, 247:1306-1310), wherein the authors indicate proteins are surprisingly tolerant of amino acid substitutions. For example, Bowie et al. disclose that there are two main approaches for studying the tolerance of a polypeptide sequence to change. The first method relies on the process of evolution, in which mutations are either accepted or rejected by natural selection. The second approach uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene and selects or screens to identify sequences that maintain functionality. As stated by the authors, these studies have revealed that proteins are surprisingly tolerant of amino acid substitutions. The authors further indicate which changes are likely to be permissive at a certain position of the protein. For example, most buried amino acid residues require non-polar side chains, whereas few features of surface side chains are generally conserved. Other such phenotypically silent substitutions are described in Bowie et al, and the references cited therein.
The present invention also includes isolated polynucleotides encoding a polypeptide of the present invention. A polynucleotide encoding an OppA polypeptide is referred to herein as an oppA polynucleotide. oppA polynucleotides may have a nucleotide sequence encoding a polypeptide having the amino acid sequence shown in SEQ ID NO:2. An example of the class of nucleotide sequences encoding such a polypeptide is the complement of SEQ ID NO:1, preferably the reverse complement of nucleotides 1-1,542 of SEQ ID NO: 1. It should be understood that a polynucleotide encoding a OppA polypeptide represented by SEQ ID NO:2 is not limited to the nucleotide sequence disclosed at the complement of SEQ ID NO:1, but also includes the class of polynucleotides encoding such polypeptides as a result of the degeneracy of the genetic code. For example, the naturally occurring complement of SEQ ID NO:1 is but one member of the class of nucleotide sequences encoding a polypeptide having the amino acid sequence SEQ ID NO:2. The class of nucleotide sequences encoding a selected polypeptide sequence is large but finite, and the nucleotide sequence of each member of the class may be readily determined by one skilled in the art by reference to the standard genetic code, wherein different nucleotide triplets (codons) are known to encode the same amino acid.
An oppA polynucleotide of the present invention may have sequence similarity with the nucleotide sequence of SEQ ID NO:1, preferably the reverse complement of nucleotides 1-1,542 of SEQ ID NO:1. oppA polynucleotides having sequence similarity with the nucleotide sequence of SEQ ID NO:1 encode an OppA polypeptide. An oppA polynucleotide may be isolated from a microbe, such as a member of the genera Pasteurellaceae, such as an H. parasuis, or may be produced using recombinant techniques, or chemically or enzymatically synthesized. An oppA polynucleotide of the present invention may further include heterologous nucleotides flanking the open reading frame encoding the oppA polynucleotide. Typically, heterologous nucleotides may be at the 5′ end of the coding region, at the 3′ end of the coding region, or the combination thereof. The number of heterologous nucleotides may be, for instance, at least 10, at least 100, or at least 1000.
Whether a polypeptide has oligopeptide permease activity may be determined by in vitro assays. Such assays are known to the person skilled in the art and are routine.
The present invention also includes fragments of the polypeptides described herein, and the polynucleotides encoding such fragments. A fragment may include antigenic epitopes that are not present on the oligopeptide permease expressed by Actinobacillus pleuropneumoniae and described at Genbank accession number ABY69417.1 (SEQ ID NO:4, which is encoded by nucleotides 948758-950332 of Genbank accession number CP000687, SEQ ID NO:3, FIG. 11). A polypeptide fragment may include a sequence of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, or at least 100 amino acid residues.
A polypeptide of the present invention or a fragment thereof may be expressed as a fusion polypeptide that includes a polypeptide of the present invention or a fragment thereof and an additional amino acid sequence. For instance, the additional amino acid sequence may be useful for purification of the fusion polypeptide by affinity chromatography. Various methods are available for the addition of such affinity purification moieties to proteins. Representative examples include, for instance, a polyhistidine-tag (His-tag) and maltose-binding protein (see, for instance, Hopp et al. (U.S. Pat. No. 4,703,004), Hopp et al. (U.S. Pat. No. 4,782,137), Sgarlato (U.S. Pat. No. 5,935,824), and Sharma (U.S. Pat. No. 5,594,115)). In another example, the additional amino acid sequence may be a carrier polypeptide. The carrier polypeptide may be used to increase the immunogenicity of the fusion polypeptide to increase production of antibodies that specifically bind to a polypeptide of the invention. The invention is not limited by the types of carrier polypeptides that may be used to create fusion polypeptides. Examples of carrier polypeptides include, but are not limited to, keyhole limpet hemacyanin, bovine serum albumin, ovalbumin, mouse serum albumin, rabbit serum albumin, and the like.
A polynucleotide of the present invention may be present in a vector. A vector is a replicating polynucleotide, such as a plasmid, phage, or cosmid, to which another polynucleotide may be attached so as to bring about the replication of the attached polynucleotide. Construction of vectors containing a polynucleotide of the invention employs standard ligation techniques known in the art. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989). A vector may provide for further cloning (amplification of the polynucleotide), i.e., a cloning vector, or for expression of the polynucleotide, i.e., an expression vector. The term vector includes, but is not limited to, plasmid vectors, viral vectors, cosmid vectors, and artificial chromosome vectors. Examples of viral vectors include, for instance, lambda phage vectors, P1 phage vectors, M13 phage vectors, adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, retroviral vectors, and herpes virus vectors. Typically, a vector is capable of replication in a microbial host, for instance, a prokaryotic bacterium, such as E. coli. Preferably the vector is a plasmid.
Selection of a vector depends upon a variety of desired characteristics in the resulting construct, such as a selection marker, vector replication rate, and the like. In some aspects, suitable host cells for cloning or expressing the vectors herein include prokaryotic cells. Suitable prokaryotic cells include eubacteria, such as gram-negative microbes, for example, E. coli. Vectors may be introduced into a host cell using methods that are known and used routinely by the skilled person. For example, calcium phosphate precipitation, electroporation, heat shock, lipofection, microinjection, and viral-mediated nucleic acid transfer are common methods for introducing nucleic acids into host cells.
Polynucleotides of the present invention may be obtained from microbes, for instance, H. parasuis. H. parasuis useful in the present invention may be obtained from pigs, preferably pigs showing signs and/or symptoms of H. parasuis disease. Accordingly, in some embodiments a useful H. parasuis is one capable of causing disease in a pig. Polynucleotides of the present invention may be produced in vitro or in vivo. For instance, methods for in vitro synthesis include, but are not limited to, chemical synthesis with a conventional DNA/RNA synthesizer. Commercial suppliers of synthetic polynucleotides and reagents for such synthesis are well known. Likewise, polypeptides of the present invention may be obtained from microbes, or produced in vitro or in vivo.
An expression vector optionally includes regulatory sequences operably linked to the coding region. The invention is not limited by the use of any particular promoter, and a wide variety of promoters are known. Promoters act as regulatory signals that bind RNA polymerase in a cell to initiate transcription of a downstream (3′ direction) coding region. The promoter used may be a constitutive or an inducible promoter. It may be, but need not be, heterologous with respect to the host cell.
An expression vector may optionally include a ribosome binding site and a start site (e.g., the codon ATG or GTG) to initiate translation of the transcribed message to produce the polypeptide. It may also include a termination sequence to end translation. A termination sequence is typically a codon for which there exists no corresponding aminoacetyl-tRNA, thus ending polypeptide synthesis. The polynucleotide used to transform the host cell may optionally further include a transcription termination sequence.
A vector introduced into a host cell optionally includes one or more marker sequences, which typically encode a molecule that inactivates or otherwise detects or is detected by a compound in the growth medium. For example, the inclusion of a marker sequence may render the transformed cell resistant to an antibiotic, or it may confer compound-specific metabolism on the transformed cell. Examples of a marker sequence are sequences that confer resistance to kanamycin, ampicillin, chloramphenicol, tetracycline, and neomycin.
The present invention also includes antibodies that specifically bind a polypeptide of the present invention. An antibody that specifically binds an OppA polypeptide of the present invention, preferably, SEQ ID NO:2 or a fragment thereof, does not bind to an oligopeptide permease expressed by Actinobacillus pleuropneumoniae and described at Genbank accession number ABY69417.1 (SEQ ID NO:4).
Antibody may be produced using a polypeptide of the present invention, or a fragment thereof. The antibody may be polyclonal or monoclonal. Laboratory methods for producing, characterizing, and optionally isolating polyclonal and monoclonal antibodies are known in the art (see, for instance, Harlow E. et al., 1988, Antibodies: A laboratory manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor). For instance, a polypeptide of the present invention may be administered to an animal, such as a mammal or a chicken, in an amount effective to cause the production of antibody specific for the administered polypeptide. Optionally, a polypeptide may be mixed with an adjuvant, for instance Freund's incomplete adjuvant, to stimulate the production of antibodies upon administration. Production of antibody that specifically binds SEQ ID NO:2 and does not specifically bind SEQ ID NO:4 may be accomplished by use of one or more fragments from SEQ ID NO:2 that have reduced identity with a series of amino acids in SEQ ID NO:4. By way of example and without intending to be limiting, fragments of SEQ ID NO:2 that include, for instance, amino acids 30 to 47, 361 to 382, or 397 to 412 (see FIG. 11), may be administered to an animal. The antibodies that result would be expected to include those specifically binding to SEQ ID NO:2 and not specifically binding to SEQ ID NO:4.
An antibody of the present invention may be produced by recombinant methods known in the art. An antibody of the present invention may be modified by recombinant means to increase efficacy of the antibody in mediating the desired function.
Antibody fragments include at least a portion of the variable region of an antibody that specifically binds to its target. Examples of antibody fragments include, for instance, scFv, Fab, F(ab′)2, Fv, a single chain variable region, and the like. Fragments of intact molecules can be generated using methods well known in the art and include enzymatic digestion and recombinant means.
Whether an antibody of the present invention specifically binds to a polypeptide of the present invention may be determined using methods known in the art. For instance, specificity may be determined by testing antibody binding to SEQ ID NO:2 and a polypeptide having the amino acid sequence described at Genbank accession number ABY69417.1 (SEQ ID NO:4).
An antibody of the present invention may be coupled (also referred to as conjugated) to a detectable label, e.g., a molecule that is easily detected by various methods. Examples include, but are not limited to, radioactive elements; enzymes (such as horseradish peroxidase, alkaline phosphatase, and the like); fluorescent, phosphorescent, and chemiluminescent dyes; latex and magnetic particles; cofactors (such as biotin); dye crystallites, gold, silver, and selenium colloidal particles; metal chelates; coenzymes; electroactive groups; oligonucleotides, stable radicals, and others. Methods for conjugating a detectable label to antibody vary with the type of label, and such methods are known and routinely used by the person skilled in the art.
Also provided herein are other molecules that specifically bind a polypeptide of the present invention. Examples of such molecules include DNA and/or RNA aptamers. Methods for making such molecules are known to the skilled person and are routine.
The present invention also includes genetically modified microbes and compositions that include genetically modified microbes. In some embodiments a genetically modified microbe has an exogenous polynucleotide encoding an OppA polypeptide described herein, or a fragment thereof. Compared to a control microbe that is not genetically modified, a genetically modified microbe may exhibit production of an OppA polypeptide or a fragment thereof. A polynucleotide encoding an OppA polypeptide may be present in the microbe as a vector or integrated into a chromosome. Examples of microbes that can be genetically modified to include an exogenous polynucleotide encoding an OppA polypeptide described herein, or a fragment thereof, include, but are not limited to, attenuated microbes such as Bacille Calmette-Guerin (BCG), Listeria monocytogenes, Salmonellae, Shigellae, and E. coli. 
In another aspect, a genetically modified microbe is an H. parasuis. The H. parasuis may include a polynucleotide encoding an OppA polypeptide (an oppA coding region), where the oppA coding region includes a mutation. The mutation may be one that results in attenuation of the microbe. An attenuated microbe is able to replicate in an animal and induce an immune response, but generally is not pathogenic and has a reduced ability to cause the clinical signs and/or symptoms of disease in an animal. An attenuated H. parasuis has a reduced ability to cause in pigs the clinical signs of pneumonia, arthritis, meningitis, pleuritis, and peritonitis. Whether a genetically modified H. parasuis is attenuated can be determined by testing using pigs, the natural host for H. parasuis, or guinea pigs, a model system recognized in the art as relevant in the study of H. parasuis pathogenesis in pigs.
Examples of mutations that can be used in the production of a genetically modified H. parasuis include point mutations, such as transition and/or transversion point mutations, deletions, and insertions. A deletion may include deletion of part or an entire nucleotide sequence encoding OppA, or deletion of a regulatory region of an oppA coding region. The genetically modified H. parasuis may encode a fragment of an OppA polypeptide or not encode an OppA polypeptide. If the genetically modified H. parasuis encodes a fragment of an OppA polypeptide, the polypeptide typically does not have oligopeptide permease activity. Typically, a mutation useful to produce a genetically modified H. parasuis for use in one of the methods described herein is stable and non-reverting.
A variety of methods that can be used to modify an oppA coding region in an H. parausis are known and used routinely by the skilled person. For instance, DNA integration cassettes (also referred to as DNA mutagenic cassettes) can be used to replace a chromosomal oppA coding region in a wild-type cell by homologous recombination. Such cassettes typically include the mutation to be inserted, homologous nucleotide sequences to target the mutation to the oppA coding region, and a marker sequence. The actual nucleotide sequence of an oppA coding region in an H. parasuis may vary slightly from a publicly available sequence; however, the actual nucleotide sequence can be easily determined using routine methods to clone the coding region and determine the nucleotide sequence. Typically, the cloning of the coding region can be accomplished by use of the known nucleotide sequence in many routine techniques including, for instance, making primers for use in a polymerase chain reaction to amplify the coding region, or making a probe to screen a library for the coding region.
The present invention also provides compositions including a genetically modified microbe, polypeptides, polynucleotides, molecules that specifically bind an OppA polypeptide described herein (such as an antibody), or combinations thereof. Such compositions may include a pharmaceutically acceptable carrier. As used herein “pharmaceutically acceptable carrier” includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration and not deleterious to a recipient thereof. A composition of the present invention may be referred to as a vaccine. The term “vaccine” as used herein refers to a composition that, upon administration to an animal, will increase the likelihood the recipient is protected against H. parasuis. For instance, when the composition includes or encodes an immunogenic polypeptide, administration to the animal typically produces an immunological response to the polypeptide and results in immunity.
The compositions of the present invention may be formulated in pharmaceutical preparations in a variety of forms adapted to the chosen route of administration, including routes suitable for stimulating an immune response to an antigen. Thus, a composition of the present invention can be administered via known routes including, for example, oral; parenteral including intradermal, transcutaneous and subcutaneous; intramuscular, intravenous, intraperitoneal, etc. and topically, such as, intranasal, intrapulmonary, intramammary, intravaginal, intrauterine, intradermal, transcutaneous and rectally, etc. It is foreseen that a composition can be administered to a mucosal surface, such as by administration to the nasal or respiratory mucosa (e.g., via a spray or aerosol), in order to stimulate mucosal immunity, such as production of secretory IgA antibodies, throughout the animal's body.
A composition may include a polypeptide described herein. Such a composition is often referred to as a subunit vaccine. The polypeptides present in the composition may be obtained by isolation or purification from an H. parasuis, or by using recombinant, enzymatic, or chemical techniques described herein, such as expression systems (e.g., expression in an E. coli host) followed by isolation or purification of the polypeptide.
A composition may include a polynucleotide of the present invention. The polynucleotide can include DNA, RNA, or a combination thereof. The polynucleotide may be supplied as part of a vector or as a “naked” polynucleotide.
The formulations may be conveniently presented in unit dosage form and may be prepared by methods well known in the art of pharmacy. Methods of preparing a composition with a pharmaceutically acceptable carrier include the step of bringing the active compound (e.g., a polypeptide or genetically modified microbe) into association with a carrier that constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing the active compound into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into the desired formulations. In general, a composition can be formulated to be compatible with its intended route of administration. More specifically, the compositions of the present invention may be administered to any tissue of an animal, including, but not limited to, muscle (such as skeletal muscle or cardiac muscle), skin, lung tissue, intestinal tissue, and the like. A composition of the present invention may be administered to any internal cavity of an animal, including, but not limited to, lungs, mouth, nasal cavity, stomach, peritoneal cavity, intestine, veins, and the like.
A composition including a pharmaceutically acceptable carrier can also include an adjuvant. An “adjuvant” refers to an agent that can act in a nonspecific manner to enhance an immune response to a particular antigen, thus potentially reducing the quantity of antigen necessary in any given immunizing composition, and/or the frequency of injection necessary in order to generate an adequate immune response to the antigen of interest. Adjuvants may include, for example, IL-1, IL-2, emulsifiers, muramyl dipeptides, dimethyl dioctadecyl ammonium bromide (DDA), pyridine, aluminum hydroxide, oils, saponins, alpha-tocopherol, polysaccharides, emulsified paraffins, ISA-70, RIBI and other substances known in the art.
In another embodiment, a composition of the invention including a pharmaceutically acceptable carrier can include a biological response modifier, such as, for example, IL-2, IL-4 and/or IL-6, TNF, IFN-alpha, IFN-gamma, and other cytokines that effect immune cells. An immunizing composition can also include other components known in the art such as antibiotics, preservatives, anti-oxidants, or chelating agents.
Any route of administration may be used so long as it results in the delivery of an amount sufficient to generate an immune response (e.g., when polypeptide, polynucleotide, or attenuated H. parasuis is administered) or a protective response (e.g., when an antibody is administered) in an animal in need of such response.
A composition of the present invention may be administered in an amount sufficient to treat certain conditions as described herein. For instance, the amount of polypeptides or genetically modified microbe present in a composition of the present invention can vary. For example, the dosage of polypeptides can be between 0.01 micrograms (μg) and 300 milligrams (mg), typically between 0.05 mg and 10 mg. When the composition is a preparation including genetically modified microbes, the cells can be present at a concentration of, for instance, at least 102 bacteria/ml, at least 103 bacteria/ml, at least 104 bacteria/ml, at least 105 bacteria/ml, at least 106 bacteria/ml, at least 107 bacteria/ml, at least 108 bacteria/ml, or at least 109 bacteria/ml. For an injectable composition (e.g. subcutaneous, intramuscular, etc.) the polypeptides may be present in the composition in an amount such that the total volume of the composition administered is 0.5 ml to 5.0 ml, typically 1.0 to 2.0 ml. When the composition is a preparation of genetically modified microbes, the cells are preferably present in the composition in an amount that the total volume of the composition administered is 0.5 ml to 5.0 ml, typically 1.0 to 2.0 ml. The amount administered will vary depending on various factors including, but not limited to, the specific polypeptides chosen, the weight, physical condition and age of the animal, and the route of administration. Thus, the absolute weight of the polypeptide included in a given unit dosage form can vary widely, and depends upon factors such as the species, age, weight and physical condition of the animal, as well as the method of administration. Such factors can be determined by one of skill in the art.
The present invention is also directed to methods of using the compositions described herein. In one aspect, the present invention provides methods for detecting antibody that specifically binds an OppA polypeptide described herein. These methods are useful in, for instance, detecting whether an animal has antibody that specifically binds polypeptides of the present invention, diagnosing whether an animal has been systemically infected by H. parasuis, or monitoring maternal antibody levels to predict when a vaccine may be given to a young animal. Such diagnostic systems may be in kit form. The methods typically include testing a biological sample obtained from an animal for the presence of antibody that specifically binds an OppA polypeptide described herein. The biological sample may be derived from the circulatory system and may be, for instance, blood (including serum), oral fluid (such as saliva), colostrum, or nasal, tracheal, bronchial, or lung lavage. The method may include contacting an antibody with a preparation that includes an OppA polypeptide described herein to result in a mixture. The method may further includes incubating the mixture under conditions to allow the antibody to specifically bind the polypeptide to a polypeptide:antibody complex. As used herein, the term “polypeptide:antibody complex” refers to the complex that results when an antibody specifically binds to a polypeptide. The preparation that includes the polypeptides of the present invention may also include reagents, for instance a buffer, that provide conditions appropriate for the formation of the polypeptide:antibody complex. The polypeptide:antibody complex is then detected.
These assays may be performed within various immunological assay formats known in the art and routinely used, including but not limited to various types of radioimmunoassays, enzyme-linked immunosorbent assays (ELISA), enzyme-linked immunofluorescent assays (ELIFA), immunoprecipitation, Western blot analysis, serum neutralization, such as standard SN and modified SN, and the like. The OppA polypeptide may be in solution or bound to a surface, such as polystyrene or microsphere. In one embodiment, an OppA polypeptide described herein may be bound to the surface of a well, and a biological sample added to the well and incubated under conditions suitable for the formation of a polypeptide:antibody complex between the bound OppA polypeptide and any anti-OppA antibody present in the biological sample. Detection of the anti-OppA antibody may be accomplished using various routine detection methods known in the art, including, for instance, antibodies that will specifically bind the polypeptide:antibody complex. Such antibodies, including anti-pig antibody, are commercially available and can be easily made. Such antibodies may be conjugated to a detectable label, such as radioactive elements; enzymes (such as horseradish peroxidase and alkaline phosphatase); fluorescent, phosphorescent, and chemiluminescent dyes; latex and magnetic particles; cofactors (such as biotin); dye crystallites, gold, silver, and selenium colloidal particles; metal chelates; coenzymes; electroactive groups; oligonucleotides, stable radicals, and others.
In one embodiment, the methods may be used to monitor maternal antibody levels to predict when a vaccine may be given to a newborn animal. Newborn animals typically have no antibodies of their own, and are not capable of producing antibody until their immune system has matured. A newborn animal obtains antibody helpful in protection from pathogens by ingesting its mother's milk (maternal colostrum) soon after birth; however, this antibody can neutralize vaccine antigens preventing stimulation of the immune system. Thus, it can be useful to know the level of maternal antibody to an antigen present before administration of that antigen. In some embodiments, the titer of maternal antibody to an OppA polypeptide described herein can be determined as described herein. Once the maternal antibody titer is known, it is possible to predict when the maternal antibody titer will drop to a level that permits vaccination with, for instance, an OppA polypeptide.
Other methods of using the compositions described herein include, for instance, methods for detecting an OppA polypeptide. These methods are useful in, but not limited to, detecting H. parasuis in an animal, detecting H. parasuis in tissues from an animal, and diagnosing a disease caused by H. parasuis (including during an infection or after an infection). Such diagnostic systems may be in kit form. The methods typically include testing a biological sample obtained from an animal for the presence of an OppA polypeptide described herein. The biological sample may be derived from the circulatory system and may be, for instance, blood (including serum), oral fluid (such as saliva), colostrum, or nasal, tracheal, bronchial, or lung lavage. The biological sample may be derived from the lymphatic system and may be, for instance, tonsil tissue. The method may include contacting an OppA polypeptide described herein with a preparation that includes an antibody to result in a mixture. The method may further include incubating the mixture under conditions to allow the antibody to specifically bind the polypeptide to form a polypeptide:antibody complex. The preparation that includes the antibody may also include reagents, for instance a buffer, that provide conditions appropriate for the formation of the polypeptide:antibody complex. The polypeptide:antibody complex is then detected.
These assays may be performed within various immunological assay formats known in the art and routinely used, including but not limited to various types of radioimmunoassays, enzyme-linked immunosorbent assays (ELISA), enzyme-linked immunofluorescent assays (ELIFA), immunocytochemical methods, immunoprecipitation, immunohistochemical analysis, Western blot analysis, serum neutralization, such as standard SN and modified SN, and the like. The antibody may be in solution or bound to a surface, such as polystyrene or microsphere. In one embodiment, an antibody that specifically binds an OppA polypeptide described herein may be bound to the surface of a well, and a biological sample added to the well and incubated under conditions suitable for the formation of a polypeptide:antibody complex between the anti-OppA antibody OppA polypeptide present in the biological sample. Detection of the polypeptide:antibody complex may be accomplished using various detection methods known in the art and used routinely, including, for instance, antibodies that will specifically bind the polypeptide:antibody complex, such as anti-OppA antibody. Such antibodies can be easily made. Such antibodies may be conjugated to a detectable label, such as radioactive elements; enzymes (such as horseradish peroxidase and alkaline phosphatase); fluorescent, phosphorescent, and chemiluminescent dyes; latex and magnetic particles; cofactors (such as biotin); dye crystallites, gold, silver, and selenium colloidal particles; metal chelates; coenzymes; electroactive groups; oligonucleotides, stable radicals, and others.
In some aspects of the invention, detecting H. parasuis includes detecting such a microbe in an animal. These methods may include providing a biological sample from an animal. In this aspect the animal is one that is suspected of harboring the microbe (presently infected), or may be a member of a group that is being screened for the presence of H. parasuis. Antibody the specifically binds OppA described herein may be mixed with the biological sample and incubated under conditions to form a complex with H. parasuis. The complex is then detected, and the presence of the complex indicates the presence of H. parasuis in the biological sample. The complex may be antibody:H. parasuis, or antibody:OppA polypeptide. The detection of antibodies is known in the art and may include, for instance, immunofluorescence and peroxidase.
Other methods for detecting a H. parasuis include the amplification of an oppA polynucleotide, such as by the polymerase chain reaction (PCR), microarrays, hybridization using probes, such as in situ hybridization, and the like. The polynucleotide may be one that is, for instance, present in a biological sample from an animal that is suspected of harboring the H. parasuis, or a member of a group that is being screened for the presence of the H. parasuis. 
The method may include contacting a biological sample that may include an oppA polynucleotide with an oligonucleotide, such as a probe for hybridization, or a primer pair for amplification. Amplification includes incubation under conditions to form a detectable amplified polynucleotide. As used herein, a “primer pair” refers to two single stranded polynucleotides that can be used together to amplify a region of a polynucleotide, preferably by a PCR. Many variations of PCR exist, including, for instance, real time PCR and mass tag PCR, and are useful herein. The polynucleotide that results from amplifying a region of a polynucleotide is referred to as an “amplification product” or an “amplified polynucleotide.”
Primers that amplify a portion of an oppA polynucleotide can be designed using readily available computer programs, such as DNAStar Lasergene (Madison, Wis.) and Primer3 (Rozen and Skaletsky, 2000, In: Krawetz and Misener (eds.), Bioinformatics Methods and Protocols: Methods in Molecular Biology. Humana Press, Totowa, N.J., pp 365-386). Factors that can be considered in designing primers include, but are not limited to, melting temperatures, primer length, size of the amplification product, and specificity. Primer length is generally between about 15 and about 30 nucleotides. The conditions for amplifying a polynucleotide by PCR vary depending on the nucleotide sequence of primers used, and methods for determining such conditions are routine in the art. An example of a primer pair that can be used to amplify an oppA coding region from H. parasuis and not amplify an oppA coding region from another microbe is 5′-CGGCTTACACCAATGAATGA (forward) (SEQ ID NO:7) and 5′-GTGTTGGTGCTGGTTTAGCA (reverse) (SEQ ID NO:8). The size of the amplified polynucleotide is 1,519 bases.
Typical formats in which polynucleotides, such as primer pairs and/or probes, may be used to detect H. parasuis include the use of polynucleotides in solution or bound to a surface, such as polystyrene or microsphere.
The methods further include methods for genotyping an H. parasuis isolated from a pig or detected in clinical samples by PCR. The genomic region analyzed when genotyping an H. parasuis includes nucleotides present in an oppA coding region. Typically, a portion of an oppA coding region is amplified and then either the nucleotide sequence of the amplified fragment is determined, or the amplified fragment is subjected to restriction fragment length polymorphism (RFLP) analysis. The sequence of the amplified fragment or the restriction fragments may be compared for strain identification based on oppA sequence type. Preferably, when the nucleotide sequence of an amplified fragment is determined, nucleotides near the 3′ end are used for strain typing (nucleotides 667-1394). For instance, when the primers described above are used for amplification of the oppA gene, the reverse primer may be used as the primer for sequencing. Thus, an oppA coding region described herein may be used as a molecular epidemiology marker for strain differentiation. Moreover, the identification of H. parasuis strains based on oppA sequencing permits quick selection of strains that can be used in the production of an autogenous vaccine.
Methods for using the compositions described herein also include administering to an animal an effective amount of a composition of the present invention, for instance an OppA polypeptide or fragment thereof, or a genetically modified microbe. The animal can be, for instance, porcine (including, for instance, swine), avian (including, for instance, chickens or turkeys), bovine (including, for instance, cattle and bison), caprine (including, for instance, goats), ovine (including, for instance, sheep), equine (including, for instance, horses), a companion animal (including, for instance, dogs or cats), members of the family Cervidae (including, for instance, deer, elk, moose, caribou and reindeer), murine (including, for instance, mice or rats), or human.
In some aspects, the methods may further include additional administrations (e.g., one or more booster administrations) of the composition to the animal to enhance or stimulate a secondary immune response. A booster can be administered at a time after the first administration, for instance, one to eight weeks, preferably two to four weeks, after the first administration of the composition. Subsequent boosters can be administered one, two, three, four, or more times annually.
In one aspect the invention is directed to treating an infection in an animal, caused by an H. parasuis. As used herein, the term “infection” refers to the presence of an H. parasuis in an animal's body and interfering with the normal functioning of the host animal.
Treating an infection can be prophylactic or, alternatively, can be initiated after the animal is infected by the microbe. Treatment that is prophylactic—e.g., initiated before a subject is infected by a microbe or while any infection remains subclinical—is referred to herein as treatment of a subject that is “at risk” of infection. As used herein, the term “at risk” refers to an animal that may or may not actually possess the described risk. Thus, typically, an animal “at risk” of infection by a microbe is an animal present in an area where animals have been identified as infected by the microbe and/or is likely to be exposed to the microbe even if the animal has not yet manifested any detectable indication of infection by the microbe and regardless of whether the animal may harbor a subclinical amount of the microbe. Accordingly, administration of a composition can be performed before, during, or after the animal has first contact with the microbe. Treatment initiated after the animal's first contact with the microbe may result in decreasing the severity of symptoms and/or clinical signs of infection by the microbe, completely removing the microbe, and/or decreasing the likelihood of experiencing a clinically evident infection compared to an animal to which the composition is not administered.
The method includes administering an effective amount of the composition of the present invention to an animal having, or at risk of having, an infection caused by an H. parasuis, and optionally determining whether the number of microbes causing the infection has decreased. In this aspect of the invention, an “effective amount” is an amount effective to reduce the number of the specified microbes in an animal or reduce the likelihood that the animal experiences a clinically-evident infection compared to an animal to which the composition is not administered. Methods for determining whether an infection is caused by an H. parasuis are routine and known in the art, as are methods for determining whether the infection has decreased. Such methods include, for instance, measuring tissues colonized by H. parasuis during infection for the presence and/or absence and/or quantity of H. parasuis. 
In another embodiment, the method includes administering an effective amount of the composition of the present invention to an animal having, or at risk of having, an infection caused by an H. parasuis, and optionally determining whether an immune response against the antigen, for instance, the OppA polypeptide or the genetically modified microbe, has occurred in the animal. In this aspect of the invention, an “effective amount” is an amount effective to result in an immune response to the administered antigen. Without intending to be limiting, this aspect of the invention may be useful to induce an immune response in a breeding female, such as a sow. An immune response in a sow against the administered antigen can be passed to the sow's offspring via maternal colostrum, resulting in the passive transfer of immunity. This is particularly advantageous with farm animals, as passive transfer of immunity generally does not occur through the placenta before birth.
In another aspect, the present invention is directed to methods for treating one or more symptoms or clinical signs of certain conditions in an animal that may be caused by infection by an H. parasuis. The method includes administering an effective amount of a composition of the present invention to an animal having or at risk of having a condition, or exhibiting symptoms and/or clinical signs of a condition, and determining whether at least one symptom and/or clinical sign of the condition is changed, preferably, reduced. Examples of conditions and/or clinical signs caused by H. parasuis include, but are not limited to, pneumonia, arthritis, meningitis, pleuritis, peritonitis, heart sac infection, fever, respiratory distress, lameness, CNS signs (e.g., lateral decumbency, paddling, and nistagmus), lameness/stiffness, slight swellings over the joints and tendons, and/or a short cough of 2-3 episodes. In swine disease caused by H. parasuis is often referred to as Glässers Disease.
Treatment of symptoms and/or clinical signs associated with these conditions can be prophylactic or, alternatively, can be initiated after the development of a condition described herein. As used herein, the term “symptom” refers to subjective evidence of disease or condition experienced by the patient and caused by infection by a microbe. As used herein, the term “clinical sign” or, simply, “sign” refers to objective evidence of disease or condition caused by infection by a microbe. Symptoms and/or clinical signs associated with conditions referred to herein and the evaluations of such symptoms are routine and known in the art. Treatment that is prophylactic, for instance, initiated before a subject manifests symptoms or signs of a condition caused by a microbe, is referred to herein as treatment of a subject that is “at risk” of developing the condition. Thus, typically, an animal “at risk” of developing a condition is an animal present in an area where animals having the condition have been diagnosed and/or is likely to be exposed to a microbe causing the condition even if the animal has not yet manifested symptoms or signs of any condition caused by the microbe. Accordingly, administration of a composition can be performed before, during; or after the occurrence of the conditions described herein. Treatment initiated after the development of a condition may result in decreasing the severity of the symptoms of one of the conditions, or completely removing the symptoms. In this aspect of the invention, an “effective amount” is an amount effective to prevent the manifestation of symptoms of a disease, decrease the severity of the symptoms of a disease, and/or completely remove the symptoms.
The present invention also provides methods for decreasing colonization by an H. parasuis, for instance blocking the attachment sites of an H. parasuis, including tissues of the skeletal system (for instance, bones, cartilage, tendons and ligaments), muscular system, (for instance, skeletal and smooth muscles), circulatory system (for instance, heart, blood vessels, capillaries and blood), nervous system (for instance, brain, spinal cord, and peripheral nerves), respiratory system (for instance, nasal cavitytrachea lungs, bronchi, bronchioceles, alveoli), digestive system (for instance, mouth, salivary glands oesophagus liver stomach large and small intestine), excretory system (for instance, kidneys, ureters, bladder and urethra), endocrine system (for instance, hypothalamus, pituitary, thyroid, pancreas and adrenal glands), reproductive system (for instance, ovaries, oviduct, uterus, vagina, mammary glands, testes, and seminal vesicles), lymphatic/immune systems (for instance, lymph, lymph nodes and vessels, tonsils, mononuclear or white blood cells, such as macrophages, neutrophils, monocytes, eosinophils, basophils, and lymphocytes, including T cells and B cells), and specific cell lineages (for instance, precursor cells, epithelial cells, stem cells), and the like. As used herein, “colonization” refers to the presence of an H. parasuis in an animal's body that is not interfering with the normal functioning of the host animal.
Decreasing colonization in an animal may be performed prophylactically or, alternatively, can be initiated after the animal is colonized by the microbe. Treatment that is prophylactic, e.g., initiated before a subject is colonized by a microbe or while any colonization remains undetected—is referred to herein as treatment of a subject that is “at risk” of colonization by the microbe. Thus, typically, an animal “at risk” of colonization by a microbe is an animal present in an area where animals have been identified as colonized by the microbe and/or is likely to be exposed to the microbe even if the animal has not yet manifested any detectable indication of colonization by the microbe. Accordingly, administration of a composition can be performed before, during, or after the animal has first contact with the microbe. Treatment initiated after the animal's first contact with the microbe may result in decreasing the extent of colonization by the microbe, completely removing the microbe, and/or decreasing the likelihood that the animal becomes colonized by the microbe compared to an animal to which the composition is not administered. Thus, the method includes administering an effective amount of a composition of the present invention to an animal colonized by, or at risk of being colonized by, an H. parasuis. In this aspect of the invention, an “effective amount” is an amount sufficient to decrease colonization of the animal by the microbe, where decreasing colonization refers to one or more of: decreasing the extent of colonization by the microbe, completely removing the microbe, and/or decreasing the likelihood that the animal becomes colonized by the microbe compared to an animal to which the composition is not administered. Methods for evaluating the colonization of an animal by a microbe are routine and known in the art.
A composition of the invention can be used to provide for active or passive immunization against bacterial infection. Generally, the composition can be administered to an animal to provide active immunization. However, the composition can also be used to induce production of immune products, such as antibodies, which can be collected from the producing animal and administered to another animal to provide passive immunity. Immune components, such as antibodies, can be collected to prepare compositions (preferably containing antibody) from serum, plasma, blood, colostrum, etc. for passive immunization therapies. Passive antibody compositions and fragments thereof, e.g., scFv, Fab, F(ab′)2 or Fv or other modified forms thereof, may be administered to a recipient in the form of serum, plasma, blood, colostrum, and the like. However, the antibodies may also be isolated from serum, plasma, blood, colostrum, and the like, using known methods for later use in a concentrated or reconstituted form. Passive immunization preparations may be particularly advantageous for the treatment of acute systemic illness, or passive immunization of young animals that failed to receive adequate levels of passive immunity through maternal colostrum. Antibodies useful for passive immunization may also be useful to conjugate to various drugs or antibiotics that could be directly targeted to bacteria expressing during a systemic or localized infection a polypeptide of the present invention.
Animal models, in particular pig models, are available for experimentally evaluating the compositions of the present invention. These mouse models are commonly accepted models for the study of disease caused by H. parasuis. 
The present invention provides kits. A kit may include a polypeptide described herein (when detecting antibody to an OppA polypeptide), an antibody described herein (for detecting the presence of an H. parasuis or an OppA polypeptide), a primer pair as described herein (when amplifying a polynucleotide), or a probe (when detecting a polynucleotide by hybridization) in a suitable packaging material in an amount sufficient for at least one assay. Optionally, other reagents such as buffers and solutions, external positive or negative controls, and the like, needed to practice the invention are also included. Instructions for use of the packaged polypeptide, antibody, or primer pair may also be included.
The kits typically include packaging material, which refers to one or more physical structures used to house the contents of the kit. The packaging material can be constructed by well-known methods, preferably to provide a sterile, contaminant-free environment. The packaging material may have a marking that indicates the contents of the kit. In addition, the kit contains instructions indicating how the materials within the kit are employed. As used herein, the term “package” refers to a solid matrix or material such as glass, plastic, paper, foil, and the like.
“Instructions” typically include a tangible expression describing the various methods of the present invention, including sample preparation conditions, detection conditions, amplification conditions, and the like.
The present invention is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.
Example 1
Twenty pigs involved in an H. parasuis outbreak were selected based on clinical signs (fever >105° F., respiratory distress, lameness, CNS signs) for necropsy and H. parasuis isolation (n=10) or for antibiotic treatment to produce convalescent sera (n=10). Whole cell proteins obtained from H. parasuis isolates recovered from diseased pigs were probed with convalescent serum using Western blot. This serum was used for western blot analysis of the whole cell protein profiles of the outbreak strains and 13 H. parasuis reference strains. Convalescent serum from these pigs strongly reacted to a protein of estimated molecular weight of 52 kDa in all H. parasuis strains tested. This protein was identified in all H. parasuis reference strains tested (FIG. 1) and in all field strains obtained from brain, pleura, and pericardium during an outbreak (FIG. 2). Pre-colostrum serum obtained from a 1 day-old piglet was used as a negative control in all Western blots.
The 52 kDa protein was not detected by western blot when we used serum collected from a piglet prior to colostrum ingestion (FIG. 3). This indicated that there was no non-specific detection of the 52 kDa protein by swine serum. Convalescent serum obtained from 10 pigs involved in an H. parasuis outbreak failed to react with a 52 kDa protein in other bacterial pathogens commonly isolated from swine including Actinobacillus porcinus, Actinobacillus indolicus, Actinobacillus minor, Actinobacillus pleuropneumoniae, Actinobacillus suis, Streptococcus suis, Bordetella bronchiseptica, Pasteurella multocida, Escherichia coli, and Salmonella choleraesuis (FIG. 4). As shown in FIG. 4, the protein was detected in a Haemophilus parasuis strain isolated from brain (BR7) and in the reference strain for serotype 5.
The 52 kDa proteins obtained from an H. parasuis outbreak and serotype 5 reference strains were sequenced and results were blasted using the NCBI database. The protein was consistently identified as Oligopeptide Permease (Opp) (more specifically the OppA) based on sequencing and molecular weight in SDS-PAGE gel (FIGS. 5 and 6). The following are the complete nucleotide and amino acid sequences for the OppA gene and protein of H. parasuis that were the closest matches to the sequences generated in this study. These sequences were used to produce and express this protein in E. coli. 
OppA Nucleotide sequence SEQ ID NO:1, Genbank accession number CP001321, nucleotides 1108151-1109734. The reverse complement of nucleotides 1-1,584 encodes SEQ ID NO:2, where GTG is the start codon:







TTATTTACGGCTTACACCAATGAATGACATACCTGTGGTTGATAATGGTT


 


TAAAGCCATCTAATGCTTTGTTACTCCAAGCTGTTGGAAGTTTACGGTGA


 


ATAATTGGATAAAGTGCTGCTTCTTCAGCAATGATGTTAATCGCTTTACC


 


CCAAATTTCACGTGCTTCTGCCGGAGTTTTCGCAGCCACCGCAGCGTCAA


 


GAAGTTTTTGAACTTCCGCATATTCAGGTGTATTTGACCAGCGGAAACGT


 


TTTTTCGGCCATACATCACCACGGTACCACCAGCTTAATAATAAGTCTAA


 


GTCGTTACCGAATACTGATGGGTCACCTGGTGCGATTAATACTTCAAAGT


 


TACCTGCATCAACACCGTCATTGTATGGTGATTGTGAAGCGATGTTTGTC


 


ACTTTTACACCAGGGATCTTGTTCCAGTCTTCAAGGATCATTTGAACAGA


 


GTCCACTACCCATTTATGTGCGGTTGAACGTGTTGTAAACTCAAGTTTAT


 


CTACACCCGCTTCTTTCAACAATGCAGCCGCTTTTTTCGGATCGAAATCA


 


TAGACTGTTGAGGCTTTCACATAGTCTGGGTGAGAGTCTTGAACATAAGA


 


TGTCGCAGGTTTTGCGTTGCCTAAGAACACCACATCAACTAATTTTTGTG


 


TATCAATTGCATATTGTAATGCTTGACGTACTTTCTTGTTGTTAAACGGT


 


GCTTTTTCACAGTTAAACATTAAGAAAATTAAGCCGAATGATTGCACTGA


 


TTCTACAGCTGCTTTACGTTTTAAGCGATCCGCATCTAAGTAAGGTACGT


 


TTTCAATCGCTTGTACACGACCTGACTCTTGTGCTGCAACACGTGCTGCG


 


TCATCAAAGAGTAAGAACCATGTCATTTTTTCAACAGTTGCAGGATATGG


 


ACCTGTGTAAGCAGGGTTTGCTTCAAATACGATACGGTCATCTTTGACTG


 


CTGAAACAAATTTATATGGACCAGTACCAGCAGGGTTTGCATCAAAGACT


 


GATTGACCTTGAGCTTCAATTAATGCTTTTGGTACTATTTTGATAATGGT


 


TAAACGTTCTTTGAATAATGCAAATGGATATTTCAATTTAAATTCAACTG


 


TTTTTTGATCCACTGCAGTGACTGATTTAATGAATGGGATGAATTGTGCA


 


AAGAGTGATTTTGTATTTGGATCTAATACACGCTCAAATGAAAACACAAC


 


ATCAGCACTGGTTACTGCTGAACCGTTATGGAATACCGCACCGTCACGTA


 


AAGTGATGCGATAAGTTACATCGTCAACTTTTTCTGGTTCTTTCGCTGCA


 


AGTGCTAGATATGGCTGGCGAGTTGCTGGGTGTAAATCAACTAAACCTTC


 


AAAAATGTGGATATTAGCCGCCATTGAAGATGCACCAGTTGAACTCATTG


 


GGTCAAAACCCGTTGAAATCGGATAAGCAATACCTGTTTCGATAGTTGAA


 


CCTTTTGCTGGTGCAGCATAAGCCTGAGAAGCAAAAGTACCGATTGAACC


 


TGAGAATGCTAAACCAGCACCAACACCTGCAACTAATTTCATAAAGCCAC


 


GGCGAGATTCATTGTGTTCAAAATGTTTAGTCAC






OppA protein sequence SEQ ID NO:2 (Genbank accession number ACL32731:








MTKHFEHNESRRGFMKLVAGVGAGLAFSGSIGTFASQAYAAPAKGSTIET


 


GIAYPISTGFDPMSSTGASSMAANIHIFEGLVDLHPATRQPYLALAAKEP


 


EKVDDVTYRITLRDGAVFHNGSAVTSADVVFSFERVLDPNTKSLFAQFIP


 


FIKSVTAVDQKTVEFKLKYPFALFKERLTIIKIVPKALIEAQGQSVFDAN


 


PAGTGPYKFVSAVKDDRIVFEANPAYTGPYPATVEKMTWFLLFDDAARVA


 


AQESGRVQAIENVPYLDADRLKRKAAVESVQSFGLIFLMFNCEKAPFNNK


 


KVRQALQYAIDTQKLVDVVFLGNAKPATSYVQDSHPDYVKASTVYDFDPK


 


KAAALLKEAGVDKLEFTTRSTAHKWVVDSVQMILEDWNKIPGVKVTNIAS


 


QSPYNDGVDAGNFEVLIAPGDPSVFGNDLDLLLSWWYRGDVWPKKRFRWS


 


NTPEYAEVQKLLDAAVAAKTPAEAREIWGKAINIIAEEAALYPIIHRKLP


 


TAWSNKALDGFKPLSTTGMSFIGVSRK






Example 2
This example details the development of a Haemophilus parasuis OppA ELISA test and validation of the new test using serum samples obtained from different swine herds. Additional serum samples have been tested and validation of the test continues. The samples reported here were carefully planned and demonstrate uses for the ELISA test. Results were evaluated based on sample to positive ratios (S/P). The conclusions were made based on the trends detected. The main conclusions obtained so far include:
Non-vaccinated, clinically healthy, colonized pigs do not develop antibodies against Haemophilus parasuis OppA;
Pigs that have survived the peak of Haemophilus parasuis-related nursery mortality develop high titers of anti-OppA IgG; and
Pigs need to be systemically exposed to Haemophilus parasuis to develop anti-OppA antibodies.
Expression and Purification of OppA
The OppA polypeptide was expressed and purified by GenScript Corp. (Piscataway, N.J.). Briefly, a coding region encoding the OppA polypeptide sequence was optimized for expression in E. coli, subcloned in-frame to three different polynucleotides, each of which resulted in a polypeptide that included an N-terminal region with a polyhistidine tag, SEQ ID NO:2, and a protease cleavage site between the N-terminal region and SEQ ID NO:2 (FIG. 13). The nucleotide sequence of the coding region optimized for expression in E. coli is shown in FIG. 14 (SEQ ID NO:9). The protease cleavage sites were ENLYFQG (SEQ ID NO:10), which is cleaved by TEV protease, and DDDDK (SEQ ID NO:11), which is cleaved by enterokinase.
The polypeptide was expressed and purified using routine methods, and the N-terminal region and tag was removed by cleavage with the appropriate protease. The polypeptide was >90% pure as estimated by Coomassie blue-stained SDS-PAGE, and the yield was 0.8 mg (0.3 mg/ml in 50 mM Tris-HCl, 300 mM NaCl, pH 8.0) tag-free polypeptide prepared from 1 liter of culture. The polypeptide was expressed as a soluble faun.
ELISA Protocol
OppA was cloned and expressed in Escherichia coli and the purified antigen was used to coat ELISA plates for detection of swine anti-Haemophilus parasuis OppA antibodies using an indirect ELISA system. Purified OppA was diluted in carbonate buffer to produce a solution with a concentration of 1 μg/ml. Each well of the ELISA plates were coated with 100 μl of carbonate buffer (15 mM NaCO3, 35 mM NaHCO3, pH 9.6) incubated overnight at 4° C. Prior to adding serum samples to the plates, non-specific binding sites were inactivated by adding 300 μl of blocking solution (PBST and 0.05% non-fat dry milk (ph 9.6)) to each well and incubating the ELISA plates for 2 h at room temperature. Following blocking of non-specific binding sites, the ELISA plates were washed with phosphate buffered saline Tween-20 (PBST: phosphate buffered saline (pH 7.4) and 0.05% Tween 20) for three consecutive times. Porcine serum samples were diluted 1:50 in 5% non-fat dry milk (NFDM) in PBST and 100 μl of this solution were added to each well on the plates. A known positive and a known negative sample based on western blot analysis were added as controls. The plates containing porcine serum samples were incubated at room temperature for 1 h. Following primary antibody binding, the ELISA plates were again washed with a PBST for three consecutive times. Once the excess of washing solution was eliminated, commercially available goat anti-swine antibodies (secondary antibodies) labeled with peroxidase were diluted at 1:100,000 in 5% NFDM in PBST and 100 μl of this solution was added to the ELISA plates. Plates containing the secondary antibody were incubated at room temperature for 1 h. After the incubation period was complete, ELISA plates were washed once again three consecutive times using PBST. Following the washing step to remove unbound secondary antibodies, 100 μl of a solution containing equal parts of TMB peroxidase substrate (3,3′,5,5′-tetramethylbenzidine at a concentration of 0.4 g/L in an organic base) and peroxidase (H202 at a concentration of 0.02% in a citric acid buffer) were added to each well for detection of bound secondary goat anti-swine antibodies. The reaction was quenched by adding 100 μl of 1 M phosphoric acid solution to each well. Optical densities obtained for each well were read at 450 nm using a microplate reader. Optical densities were used to calculate sample to positive (S/P) ratios for each sample tested.
Testing of Field Serum Samples
Convalescent Pigs:
The same serum samples that were used to discover and characterize the antigenicity of the Haemophlus parasuis OppA were tested using the developed OppA ELISA test. Convalescent serum samples were collected from 10 pigs that had survived a Haemophilus parasuis outbreak. The S/P ratios obtained for convalescent pigs varied from 0.21 to 1.2 (FIG. 7). All convalescent pigs had antibodies against the Haemophilus parasuis OppA antigen, which was in agreement with previously obtained western blot results. Eight out of 10 convalescent pigs had S/P ratios above 0.4 (FIG. 7). These results indicate that pigs that have survived a Haemophilus parasuis outbreak and had a systemic contact with the agent respond serologically to the OppA protein with production of anti-OppA IgG.
Decay of Maternal Immunity:
In order to evaluate the decay of Haemophilus parasuis-related maternal immunity, serum samples were collected from piglets at 5 and 19 days of age. At 5 days of age, S/P ratios varied from 0.32 to 0.61 (FIG. 8A), indicating the presence of maternally-derived anti-OppA antibodies. At 19 days of age, maternal antibodies were substantially reduced, with S/P ratios varying from 0.04 to 0.26 (FIG. 8B). These results demonstrate the use of the Haemophilus parasuis OppA ELISA to monitor decay of maternal immunity in young piglets. This information is useful to identify the window of susceptibility between decay of maternal immunity and development of active immune response. Furthermore, monitoring of decay of maternal antibodies can be used for strategic placement of piglet vaccination. Tracking of maternal immunity is also useful to evaluate the efficacy of sow vaccination.
Serum Profiling of a Naturally Exposed, Non-Vaccinated Population (Cross-Sectional Sampling):
Serum samples were collected from pigs at 3 and 8 weeks of age. These pigs were colonized with Haemophilus parasuis and were experiencing Haemophilus parasuis-related mortality at 5 to 6 weeks of age. Pigs were not vaccinated at the time of sampling and were obtained from non-vaccinated sows. The S/P ratios obtained for 3 week old pigs varied from −0.03 to 0.05, indicating that 3 week old pigs lacked anti-OppA antibodies (FIG. 9A) even in the presence of colonization. The S/P ratios obtained for 8 week old pigs (2-3 weeks after the peack of nursery mortality) varied from 0.12 to 0.54, indicating that pigs developed anti-OppA antibodies after they went through infection and survive the peak of Haemophilus parasuis-related mortality (FIG. 9B). These results demonstrate the use of the Haemophilus parasuis OppA ELISA to detect sysemic contact with Haemophilus parasuis in the absence of vaccination. Clinically healthy colonized pigs do not develop antibodies against Haemophilus parasuis OppA.
Serum Profiling of a Naturally Exposed, Non-Vaccinated Population (Longitudinal Sampling):
Serum samples were collected from 26 pigs at 3 and 8 weeks of age. Pigs were not vaccinated at time of sampling and were obtained from non-vaccinated gilts. The S/P ratios obtained for 3 week old pigs varied from −0.04 to 0.42, indicating that 1 week old pigs had low levels of anti-OppA maternal antibodies in this population (FIG. 10A). The S/P ratios obtained for 9 week old pigs (2-3 weeks after the peak of nursery mortality) varied from 0.09 to 0.67, indicating that pigs developed anti-OppA antibodies after they went through infection and survived the peak of Haemophilus parasuis-related mortality (FIG. 10B). These results further demonstrate the use of the Haemophilus parasuis OppA ELISA to detect systemic contact with Haemophilus parasuis in the absence of vaccination.
Example 3
Experimental Design:
Conventional healthy pigs are randomly allocated into 2 groups. For instance, twenty-two 3-week old conventional healthy pigs are randomly allocated into 2 rooms with 11 pigs per group (Table 1). Group 1 is vaccinated intramuscularly with the OppA subunit vaccine on days 0 and 14 of the experiment. Group 2 is vaccinated with adjuvant only and will remain as the non-vaccinated control. All pigs are challenged with a serotype 5 H. parasuis strain at the day 28 of the experiment and necropsied at day 35. The sample size used is adequate to detect significant differences between the proportion of affected pigs in the unvaccinated group (expected 80%) and vaccinated group (expected 20%) with 95% confidence and 80% power (Sergeant, ESG, 2009. Epitools epidemiological calculators. AusVet Animal Health Services and Australian Biosecurity Cooperative Research Centre for Emerging Infectious Disease. Available online at the AusVet Animal Health Services internet site).
All pigs are bled before and after vaccination. For instance, pigs may be bled on days 0 (prior to 1st vaccination), 14 (prior to 2nd vaccination), 28 (prior to challenge) and 35 (survivals after challenge). Serum samples are tested for PRRS virus by PCR on day 0. Clinical signs, including rectal temperature, are recorded at arrival, prior to challenge, and following challenge, for instance, weekly prior to challenge, and daily following challenge. Nasal swabs to detect colonization are collected and tested by PCR before and after vaccination, for instance, on days 0 (baseline) and 28 (effect of vaccination on clearance of H. parasuis from tonsils). Lesions observed at necropsy are recorded and graded according to severity. Thoracic and peritoneal serous surfaces are tested for the presence of H. parasuis by isolation and PCR.







TABLE 1







Example of experimental design for vaccination and challenge.












Group
Number of pigs
Vaccination
Challenge







1
11
Yes
Serotype 5



2
11
No
Serotype 5










Vaccination: Highly purified (>90%) H. parasuis OppA is used as antigen. A prototype vaccine is produced using an OppA concentration of, for instance, 200 μg/ml in oil-in-water adjuvant (Triagen®—currently used to produce autogenous H. parasuis vaccines). Pigs are vaccinated twice (for instance, at days 0 and 14) by the intramuscular route using a 1 ml dose (current route and volume used for most swine vaccines).

Challenge: Haemophilus parasuis serotype 5, ahighly virulent reference strain among U.S. swine herds, is used for challenge (Table 1). The serotype 5 reference strain has been originally isolated from the brain of a pig with systemic H. parasuis infection and carries virulence-associated genes based on VtaA multiplex PCR (Olvera et al., 2010, Vet. Res., 5-6; 41(3):26). The challenge strain is passaged onto 10 days embryonated chicken eggs prior to challenge. This method has been shown to mimic in vivo conditions and increase capsule production, which is also a virulence factor (Oliveira, 2003, Haemophilus parasuis: epidemiology, diagnosis and control. Saint Paul: University of Minnesota, Ph. D. thesis). Pigs are challenged intravenously (6 pigs per group) and intranasaally (5 pigs per group) at day 28 of the experiment with 1 ml of pure culture in exponential growth phase (Hoffmann et al., 2002, Dtsch. Tierarztl. Wochenschr., 109(6):271-276). Following challenge, pigs are monitored closely and euthanized as soon as evident clinical disease is observed (fever, respiratory distress, lameness, and/or CNS signs).
Serological testing: Serum samples obtained at days 0, 14, 28, and 35 are tested by the OppA ELISA test described in Example 2. Seroconversion will be evaluated by comparing S/P ratios before and after vaccination.
Statistical analysis: An affected pig is defined as a pig showing clinical signs (CNS signs, respiratory distress, and/or lameness), fever (temperature over 105° F.) and/or gross lesions of serositis. Because of the small sample size, non-parametric statistics are used. Specifically, Fisher's exact test is used to compare categorical data (such as mortality, proportion of affected pigs, rate of isolation of H. parasuis) between vaccinated and unvaccinated groups. Mann-Whitney U test is used to compare continuous data (distribution of ELISA s/p values).
The complete disclosure of all patents, patent applications, and publications, and electronically available material (including, for instance, nucleotide sequence submissions in, e.g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference in their entirety. Supplementary materials referenced in publications (such as supplementary tables, supplementary figures, supplementary materials and methods, and/or supplementary experimental data) are likewise incorporated by reference in their entirety. In the event that any inconsistency exists between the disclosure of the present application and the disclosure(s) of any document incorporated herein by reference, the disclosure of the present application shall govern. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.
Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.
All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

## Claims

1. A method for detecting the presence of an antibody against Haemophilus parasuis in a biological sample from an animal, comprising:
mixing the biological sample from the animal with a polypeptide under conditions suitable for formation of an antigen: antibody complex, said biological sample containing an antibody against Haemophilus parasuis, wherein the polypeptide comprises an amino acid sequence having at least 80% identity with SEQ ID NO:2, wherein the antibody binds to an oligopeptide permease OppA of Haemophilus parasuis as set forth in SEQ ID NO:2, wherein the polypeptide has oligopeptide permease activity, wherein the animal is selected from the group consisting of porcine, avian, bovine, caprine, ovine, equine, murine, human, and a companion animal; and
detecting the complex, wherein the presence of the complex indicates the animal has antibody that specifically binds to the polypeptide.

2. The method of claim 1 wherein the presence of the complex indicates the animal has been infected with Haemophilus parasuis.

3. The method of claim 1 wherein the biological sample comprises serum, oral fluid, colostrum, lung lavage, bronchial lavage, tracheal lavage, or nasal lavage.

4. The method of claim 1 wherein the polypeptide is bound to a surface.

5. The method of claim 2 wherein the H. parasuis is a serotype chosen from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15.

6. The method of claim 2 wherein the H. parasuis is a non-typable serotype.

7. The method of claim 1 wherein the antibody present in the biological sample is a first antibody and the complex is a first complex, and wherein the detecting comprises contacting the first antibody with a second antibody that specifically binds the first antibody and forms a second complex, and detecting the second complex.

8. The method of claim 1 wherein the polypeptide comprises an amino acid sequence having at least 90% identity with SEQ ID NO:2.

9. A method for detecting the presence of an antibody against Haemophilus parasuis in a biological sample from an animal, comprising:
mixing the biological sample from the animal with a polypeptide under conditions suitable for formation of an antigen: antibody complex, said biological sample containing an antibody against Haemophilus parasuis, wherein the polypeptide comprises an amino acid sequence having at least 80% identity with SEQ ID NO:2, wherein the antibody detected is an antibody that binds to an oligopeptide permease OppA of Haemophilus parasuis as set forth in SEQ ID NO:2, and does not bind to an oligopeptide permease of Actinobacillus pleuropneumoniae as set forth in SEQ ID NO:4 wherein the animal is selected from the group consisting of porcine, avian, bovine, caprine, ovine, equine, murine, human, and a companion animal; and
detecting the complex, wherein the presence of the complex indicates the animal has antibody that specifically binds to the polypeptide.